The first chapel has a dramatic altarpiece of St. Michael the Archangel (c.1635) by Guido Reni, and Gherardo delle Notti's Christ Mocked. The second chapel has a Transfiguration by Mario Balassi, and a Nativity (c. 1632) by Lanfranco. The third chapel has a Saint Francis receives stigmata by Domenichino. The fourth chapel houses a Prayer in the Gesthemane (c. 1632) by Baccio Ciarpi. In the fifth chapel is a Saint Anthony by Sacchi, who also painted the Apparition of the Virgin (1645) to Saint Bonaventure in the fifth chapel on the left. The tomb monument for Alexander Sobieski was sculpted by Camillo Rusconi. The third chapel has a Deposition by Andrea Camassei and a Stigmatization of Saint Francis (c. 1570) by Girolamo Muziano. The second chapel has a Santa Felice da Cantalice by Alessandro Turchi, while the first has a painting of Saint Paul restores vision (c. 1631) by Pietro da Cortona.

The crypt is located just under the church. Cardinal Antonio Barberini, who was a member of the Capuchin order, in 1631 ordered the remains of thousands of Capuchin friars exhumed and transferred from the friary Via dei Lucchesi to the crypt. The bones were arranged along the walls, and the friars began to bury their own dead here, as well as the bodies of poor Romans, whose tomb was under the floor of the present Mass chapel. Here the Capuchins would come to pray and reflect each evening before retiring for the night.

The crypt, or ossuary, now contains the remains of 4,000 friars buried between 1500 and 1870, during which time the Roman Catholic Church permitted burial in and under churches. The underground crypt is divided into five chapels, lit only by dim natural light seeping in through cracks, and small fluorescent lamps. The crypt walls are decorated with the remains in elaborate fashion, making this crypt a macabre work of art. Some of the skeletons are intact and draped with Franciscan habits, but for the most part, individual bones are used to create elaborate ornamental designs.

The crypt originated at a period of a rich and creative cult for their dead; great spiritual masters meditated and preached with a skull in hand.

A plaque in one of the chapels reads, in three languages, "What you are now, we once were; what we are now, you shall be." This is a memento mori.

Several renowned authors visited the crypt and left descriptions. The Marquis de Sade, who visited the crypt in 1775, wrote, “I have never seen anything more striking” (Voyage d'Italie, p. 106 of the Maurice Lever edition). Mark Twain visited the crypt in the summer of 1867, and begins Chapter XXVIII of The Innocents Abroad with 5 pages of his observations. Nathaniel Hawthorne describes the crypt in his novel The Marble Faun. Additional descriptions were written by authors Tom Weil (1992), Folke Henschen (1965) and Anneli Rufus (1999). See Christine Quigley, Skulls and Skeletons, pp. 175–176.